>Hi Frederik
>>One thing to be careful of is that last time i looked the Clontech
>monoclonal GAL4 Ab didn't detect the fusion proteins produced from their
>original 2 hybrid system, only the higher expression system 2. However,
>things might have changed, and it sounds like you're using a more
>sensitive chemiluminescent system anyway (and you might be using the
>higher expression system). In which case, the one thing i'd say is you
>seem to be diluting your antibodies an awful lot - i tend to use 1:1000
>for my primary and 1:10000 for the secondary.
>Cheers,
>Joe Boutell
>>>>boernke at IPK-Gatersleben.de (Frederik Boernke) wrote:
>>Hi!
>>>>I am currently trying to detect the GAL4 fusion protein for my 2hybrid
>>screen. Therefor I use a Clontech monoclonal mouse Ab and Pierce
>>Super Signal Ultra Chemiluminescent Substrate
>>together with an Amersham biotin/streptavidin system.
>>The samples were prepared after the method of Printen and Spraque
>>(1994) and 20 ul were loaded onto a 15% mini gel. The primary Ab was
>>dilutet 1 : 10000, secondary 1: 100000 and the strepavidin-HRP also
>>1 : 100000. When I develop the blot the only thing I see is one major
>>band at about 50 - 50 kDa ( time of exposure: 10 sec). My question now
>>is, at which step should I try some optimization. Less Protein or higher
>>dilutions of Ab's.
>>>>Thanks in Advance,
>>Ricky
>>>>>>******************************************************************
>>>>Frederik Boernke
>>Research Group Molecular Plant Physiologie
>>Institute for Plant Genetics and Crop Plant Research (IPK)
>>Corrensstr. 3
>>06466 Gatersleben
>>Tel. 039482 -5 321
>>Fax. 039482 -5 515
>>e-mail: boernke at ipk-gatersleben.de>>http://www.ipk-gatersleben.de>>>>Hey Joe,
you seem to have some time writing e-mails, that's good!
I'm actually using a Stratagene expression system, which might
express to low. About the chemilumenescence stuff: I phoned the
tech service and they told me you can easily use dilutions of 1:1000000,
hard to believe, isn't (I didn't).
Do you think there is another way to verify expression of my fusion
protein or isn't really neccesarry, because I already sequenced the
construct and recognized the proper insert that way. Should I just
carry on screening the library?
Ricky
******************************************************************
Frederik Boernke
Research Group Molecular Plant Physiologie
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel. 039482 -5 321
Fax. 039482 -5 515
e-mail: boernke at ipk-gatersleben.dehttp://www.ipk-gatersleben.de